Intensive Microalgal Cultivation and Tertiary Phosphorus Recovery from Wastewaters via the EcoRecover Process

Mixed community microalgal wastewater treatment technologies have the potential to advance the limits of technology for biological nutrient recovery while producing a renewable carbon feedstock, but a deeper understanding of their performance is required for system optimization and control. In this study, we characterized the performance of a 568 m3·day–1 Clearas EcoRecover system for tertiary phosphorus removal (and recovery as biomass) at an operating water resource recovery facility (WRRF). The process consists of a (dark) mix tank, photobioreactors (PBRs), and a membrane tank with ultrafiltration membranes for the separation of hydraulic and solids residence times. Through continuous online monitoring, long-term on-site monitoring, and on-site batch experiments, we demonstrate (i) the importance of carbohydrate storage in PBRs to support phosphorus uptake under dark conditions in the mix tank and (ii) the potential for polyphosphate accumulation in the mixed algal communities. Over a 3-month winter period with limited outside influences (e.g., no major upstream process changes), the effluent total phosphorus (TP) concentration was 0.03 ± 0.03 mg-P·L–1 (0.01 ± 0.02 mg-P·L–1 orthophosphate). Core microbial community taxa included Chlorella spp., Scenedesmus spp., and Monoraphidium spp., and key indicators of stable performance included near-neutral pH, sufficient alkalinity, and a diel rhythm in dissolved oxygen.


List of Tables
Measurement units, instrument model and manufacturer information, frequency of data collection, and sensor location for measurements made across the EcoRecover system with online sensors and analyzers.Affiliated with Figure S1.Table S2.Determination of the MDL for orthophosphate and total phosphorus via Hach TNT843 Table S3.Results from PO4-P IC MDL work: std dev of lowest 3 standards and 0.5 mg/L PO4-P, slope and y-intercept of calibration curve, R 2 value, etc. Table S4.EcoRecover mix tank and PBR biomass phosphorus content metrics.Table S5.Criteria for sample selection for SEM-EDS analysis and key system operating conditions and performance during the sampling date.S1.               Figure S18.The EcoRecover biomass N:P mass ratio had a negative linear correlation with the system SRT during a 3-month period of excellent performance.Figure S28.Biomass carbohydrate:protein ratio in the full-scale Mix tank and PBR effluent, and the difference between the PBR and mix tank biomass carbohydrate:protein ratio during November 1, 2022 to February 14, 2023.

List of Equations
Equation S1.Propagation of error for subtraction of Mix tank and PBR duplicate analyses.Table S1.Measurement units, instrument model and manufacturer information, frequency of data collection, and sensor location for measurements made across the full-scale EcoRecover system with online sensors and analyzers.

S2. Calibration and determination of MRL and MDL
Table S2.Determination of the MDL and MRL for orthophosphate and total phosphorus via Hach TNT843 for nine replicates each of 0.01 mg-P•L -1 as PO4 3-.The method reporting limit (MRL) was 0.014 and 0.016 mg-P•L -1 as PO4 3-, total phosphorus and orthophosphate, respectively.The MDL was calculated as the standard deviation of nine replicates (n = 9) multiplied by the t-value for n -1 samples (t-value = 2.896).
The MRL was 3-times the MDL. .Calibration curve for PO4 3--P concentration (mg-P•L -1 as PO4 3-) and peak area (µS•min) as measured on a Dionex ICS2100 anion ion chromatograph with a matrix background.Matrix was prepared from bench-scale PBR experiments, which were initiated with mix tank effluent from the full-scale system.Background matrix samples with no detectable orthophosphate (ICS200 signal of 0.00 µS•min) were acquired by filtering bench-scale PBR samples through 0.2 µm nitrocellulose filters after 300 minutes and 360 minutes of batch operation (orthophosphate was no longer detectable after 40 minutes of batch operation).Potassium orthophosphate was spiked into the background matrix samples to achieve final concentrations of 0.017, 0.032, 0.065, 0.13, and 0.65 mg-P•L -1 orthophosphate to generate the standard curve.
Table S3.PO4 3--P standard concentration (mg-P•L -1 as PO4 3-), peak area (µS•min), retention time (min), calculated PO4 3--P concentration (mg-P•L -1 as PO4 3-), standard deviation, MDL, and MRL for nine replicates each of the lowest four standards on the calibration curve in Figure S3 as measured on a Dionex ICS2100 anion ion chromatograph with a matrix background (as in Figure S3).From the lowest 4 points (0.017, 0.032, 0.065, and 0.13 mg-P•L -1 as PO4 3-) on the calibration curve, the average method reporting limit (MRL) was 0.027 mg-P•L -1 as PO4 3-and the MRL range was 0.022 to 0.037 mg-P•L -1 as PO4 3-.The MRL was calculated as three times the method detection limit (MDL), which was the standard deviation of nine replicates (n = 9) multiplied by the t-value for n -1 samples.S4).

S4.2. Sample selection for SEM-EDS
To understand the potential phosphorus removal mechanisms across the range of EcoRecover operating conditions and system solids phosphorus contents, a total of 12 samples were analyzed via SEM-EDS.Samples for SEM-EDS analysis were selected to represent extremes in biomass %P across the entire sampling period, biomass from periods with and without coagulant use, and start and end points in batch experiments (Table S5).

S15
During periods of coagulant use, phosphorus measured on the solids surface was generally less than that measured on solids during periods without coagulant use (Table S6).Granules, enriched with phosphorus and metals, were only present in the solids samples during periods of coagulant use or immediately after cessation of coagulant use (Figure S13).

S5. Flow imaging microscopy
To prepare aqueous samples for processing, 1 mL aliquots were diluted in deionized water by a factor of 4-6x.Dilution rate was estimated based on the previous day's measurements and by visual inspection.Diluted samples were loaded into the intake syringe port of a FlowCam 5000 flow imaging microscope (Yokogawa Fluid Imaging Technologies, Inc.).In FlowCam's VisualSpreadsheet 5 software, runs were configured to process 0.5 milliliters of sample volume.
Once a run was completed, background objects that were not automatically ignored during sample capture were manually removed.Background objects refer to particles that appear in every digital image frame due to adhesion to the walls of the flow chamber, and thus appear as thousands of identical duplicates in sample results.Duplicates were identified as particles of similar appearance with identical or similar x-y coordinates.Out of focus particles were then removed from the run data with a feature-based filter.A threshold cutoff was set for the edgegradient and transparency particle features to distinguish out of focus particles.After background and out of focus particle screening, run data was saved to disk.
For aggregate time series analysis, particle feature data were provided as input to a neural network-based classification model.The classification model generated per-particle taxonomic class predictions, which were aggregated to estimate daily total abundance of microalgal populations.To develop this classification model, training datasets were first manually annotated into image libraries corresponding to each of the dominant taxonomic groups identified across multiple sample points.Each class consisted of 4,000 to 8,000 representative particle images.
Using the deep learning library TensorFlow (version 2.5.0,Python 3.9, Apache License 2.0), a bespoke neural network was created from randomized starting weights.The model's architecture consisted of: an input layer sized to the total width of the FlowCam-exported particle feature data tables, a series of fully connected hidden layers, and an output layer whose size corresponded to the number of taxonomic classes to be predicted.Training data class sizes were increased, and model architectures and hyperparameters were adjusted until training yielded a classification model achieving > 90% overall accuracy with inter-class accuracy variability less than 10% (i.e., all classes between 85% and 95% accuracy).The model architecture and weights of this model were saved to disk and the same model was used to predict taxonomic identities of all particles for the full time series.

S6. Susceptibility to process upsets
Periods of process upset were characterized by prolonged exceedance of the 0.04 mg-P•L -1 permit.

S6.1. Upstream treatment process changes and altered EcoRecover influent quality:
On June 7, 2022, modifications were made to the SBRs upstream of the EcoRecover system that rapidly increased the TP and NO3 -and decreased the NH4 + EcoRecover system influent concentrations.The system biomass concentrations (300 to 400 mg•L -1 TSS) were insufficient to treat the high SBR effluent nutrient concentrations; without sufficient treatment capacity, the EcoRecover system effluent TP exceeded 0.

S6.2. Quaternary ammonium inhibition:
Process upsets alerted the EcoRecover operators to a potential inhibitory compound in the influent.Beginning on January 26, 2022, an external laboratory, Badger Laboratories Inc.
(Wisconsin, USA), quantified a panel of possible inhibitory compounds in the EcoRecover influent, including quaternary ammonium (QA).QA concentrations were unusually high, up to 2.5 ppm.Quantification of QA continued on site; between January 26, 2022 and July 7, 2023, the average EcoRecover influent QA concentration was 0.7 ± 0.2 ppm (Quartile 1: 0.5 ppm, Quartile 2: 0.6 ppm, Quartile 3: 0.8 ppm, Quartile 4: 2.5 ppm).Concentrations of eleven QA compounds have been reported to be inhibitory (EC50) to C. vulgaris between 0.11 to 0.20 ppm, 1-3 and 0.96 to 2.63 ppm dodecylethyldimethyl-ammonium bromide (DEAB) was reported as inhibitory to S. intermedius and D. chlorelloides. 4 Culture sensitivity to QA is a challenge to the EcoRecover system performance that has been addressed by adding QuatKill (ChemTreat, Inc., Virginia, USA), a solution designed to neutralize cationic biocides, to the influent.

S7. Bench-scale batch experiments
In the PBR batch experiments, 30.39 ± 0.13 mg-N•L -1 NH4 + was removed, with some removal due to nitrification which increased the concentrations of nitrate by 6.8 ± 1.5 mg-N•L -1 and nitrite by 0.19 ± 0.07 mg-N•L -1 .In the Mix tank experiments, 4.7 ± 0.1 mg-N•L -1 NH4 + was removed and nitrification increased the concentrations of nitrate and nitrite by 3.0 ± 0.5 mg-N•L -1 and 1.2 ± 0.4 mg-N•L -1 , respectively, which demonstrates NH4 + removal almost solely through nitrification.In the PBR batch experiments, 23% of the initial NH4 + was removed through nitrification and the remaining majority was removed through biomass assimilation.

Figure S2 .
Figure S2.Schematic of sensor and analyzer locations throughout the EcoRecover system at the Village of Roberts Wastewater Treatment Plant.Affiliated with TableS1.

Figure S3 .
Figure S3.Calibration curve for orthophosphate as measured on a Dionex ICS2100 anion ion chromatograph with a matrix background.

Figure S4 .
Figure S4.Calibration curve for NO3 --N and peak area as measured on a Dionex ICS2100 anion ion chromatograph.

Figure S5 .
Figure S5.Calibration curve for NO2 --N and peak area as measured on a Dionex ICS2100 anion ion chromatograph.

Figure S6 .
Figure S6.Orthophosphate and total phosphorus concentrations in the influent and effluent of the EcoRecover system for the period between November 1, 2022, to February 14, 2023.

Figure
Figure S7.NH4 + -N concentration in the EcoRecover system influent (secondary effluent) and permeate for the period of November 1, 2022 to February 14, 2023.

Figure
Figure S8.NO3 --N concentration in the EcoRecover system influent (secondary effluent) and permeate for the period of November 1, 2022 to February 14, 2023.

Figure S10 .
Figure S10.Non-metric multi-dimensional scaling plot showing the spatial distances among eukaryotic communities throughout the focus period.

Figure S11 .
Figure S11.Dissolved oxygen concentrations in the mix tank and photobioreactor effluent for the period of November 1, 2022 to February 14, 2023.

Figure S12 .
Figure S12.Photosynthetically active radiation (PAR) at four locations on the photobioreactors for the period of November 1, 2022 to February 14, 2023.

Figure S13 .
Figure S13.Biomass phosphorus content in the EcoRecover mix tank and PBR effluent mixed microalgal culture from February 21, 2022 to May 12, 2023.

Figure
Figure S14.SEM-EDS images of lyophilized and ground EcoRecover PBR biomass during a period of coagulant use.

Figure
Figure S15.SEM-EDS images of lyophilized and ground EcoRecover PBR biomass during 4week period without coagulant use.

Figure S16 .
Figure S16.Phosphorus and metals (sum of Na, Mg, Al, K, Ca) weight percentages of analyzed granules and biomass based on SEM-EDS.

Figure S17 .
Figure S17.Biomass N:P mass ratio for the period between November 1, 2022 to February 14, 2023.

Figure S19 .
Figure S19.Comparison of EcoRecover culture during and after a process upset.

Figure S20 .
Figure S20.Average NH4 + -N removal over time in batch, bench-scale mix tank and PBR duplicate experiments Figure S21.Average NO3 --N and NO2 --N concentrations over time in batch, bench-scale mix tank and PBR duplicate experiments.

Figure
Figure S22.Carbon:nitrogen (C:N) mass ratio for bench-scale experiments that replicated the conditions of the mix tank and PBRs.

Figure S23 .
Figure S23.Carbon:phosphorus (C:P) mass ratio for bench-scale experiments that replicated the conditions of the mix tank and PBRs.

Figure S24 .
Figure S24.Volatile suspended solids (VSS) concentration for bench-scale experiments that replicated the conditions of the mix tank and PBRs.

Figure S25 .
Figure S25.Dissolved oxygen concentrations over time for bench-scale experiments that replicated the conditions of the mix tank and PBRs.

Figure S26 .
Figure S26.Crude lipid to protein mass ratio for bench-scale experiments that replicated the conditions of the mix tank and PBRs.

Figure S27 .
Figure S27.Nitrogen:phosphorus (N:P) mass ratio for bench-scale experiments that replicated the conditions of the mix tank and PBRs.

Figure S1 .Figure S2 .
Figure S1.Photographs of the Village of Roberts EcoRecover system, including (A) the exterior of the plant showing the mix tank, CO2 storage tank, equipment building, and greenhouse (left to right), (B) the PBR fences in the interior of the greenhouse, (C) the greenhouse lit by supplemental lighting at night, (D) the system block pumps and housing for the membrane tanks and return tank, and (E) the mix tank located externally to the equipment building.

Figure S9 .Figure S10 .
Figure S9.Difference in oxidized nitrogen species (NO3 --N and NO2 --N) concentrations in EcoRecover effluent and influent 24-h composite samples for the period of November 1, 2022 to February 14, 2023.Values above the zero indicate that the rate of increase in oxidized nitrogen species, due to nitrification, was greater than the consumption of oxidized nitrogen species by the mixed microalgal community.From December 8, 2022 to February 14, 2023, nitrification (and incomplete nitrification) was negligible; the difference between effluent and influent oxidized nitrogen species was -1.0 ± 1.8 mg-N•L -1 NO3 -and 0.08 ± 0.15 mg-N•L -1 NO2 -.

Figure S11 .
Figure S11.Dissolved oxygen (DO) concentration 180-minute average in the Mix tank effluent and PBR effluent for the period between November 1, 2022 to February 14, 2023 as measured by IQ SensorNet FDO Optical Dissolved Oxygen Probes (YSI Inc.).

Figure S14 .
Figure S14.SEM-EDS images of lyophilized and ground EcoRecover PBR solids from February 13, 2023 (left) and April 5, 2022 (right), during a period of coagulant use.Bright particles represent highly localized elevated concentrations of metals and phosphorus.The composition of the granules within the EPS could not be determined due to interference from the carbon spectra but had elevated levels of metals and P.

Figure S16 .
Figure S16.Phosphorus and metals (sum of Na, Mg, Al, K, Ca) weight percentages of analyzed granules (purple) and biomass without bright particles (green) based on SEM-EDS.The exact composition of the granules could not be determined due to interference from the spectra, but granules had elevated levels of phosphorus and metals relative to the rest of the analyzed biomass.

Figure S17 .
Figure S17.Solids N:P mass ratio for the period between November 1, 2022 to February 14, 2023.Error bars represent minimum and maximum values from duplicate CHN analyses; phosphorus analyses were done in single samples.

Figure S18 .
Figure S18.The EcoRecover solids N:P mass ratio had a negative linear correlation with the system SRT (slope: -0.71, y-intercept: 5.1, R 2 = 0.63) during a 3-month period of excellent performance(November 15,  2022 to February 14, 2023).Error bars represent minimum and maximum values from duplicate CHN analyses; phosphorus analyses were done in single samples.
04 mg-P•L -1 and dramatically increased beginning on June 7, 2022, to peak on July 1 and 2, 2022 at 4.4 mg-P•L -1 until concentrations below 0.04 mg-P•L -1 were again achieved on July 30, 2022.During the upset, flow was rerouted to bypass the EcoRecover system.75% reduction in flow through the PBRs in a greenhouse in July caused the culture temperatures to reach 90º to 95° F on July 9 and 10, 2022, further delaying performance recovery (SI FigureS18A, unhealthy culture).Full flow was restored to the EcoRecover system on July 11, 2022.With gradual increase in alum addition to the WWTF headworks, EcoRecover influent TP concentrations gradually decreased to manageable levels (<1 mg•L -1 ) by the week of July 25, 2022 (SI FigureS18B, healthy culture).

Figure S19 .
Figure S19.(A) EcoRecover PBR fence from the week of July 11, 2022 after system upset and high temperature exposure; stressed culture with poor phosphorus removal flocculates and attaches to the glass.(B) EcoRecover PBR fences from on-site greenhouse camera on July 20, 2022 showing greener, denser culture.

Figure S20 .
Figure S20.Average NH4 + -N removal over time in batch, bench-scale mix tank and PBR duplicate experiments.Error bars represent minimum and maximum values from duplicate analyses.

Figure S21 .Figure S22 .
Figure S21.Average NO3 --N and NO2 --N concentrations over time in batch, bench-scale mix tank and PBR duplicate experiments.Error bars represent minimum and maximum values from duplicate analyses.

Figure S23 .Figure S24 .
Figure S23.Carbon:phosphorus (C:P) mass ratio for bench-scale experiments that replicated the conditions of the mix tank and PBRs.Error bars represent minimum and maximum values from duplicate analyses.

Figure S25 .Figure S26 .
Figure S25.Dissolved oxygen concentrations over time for bench-scale experiments that replicated the conditions of the mix tank and PBRs.Error bars represent minimum and maximum values from duplicate analyses.

Figure S27 .
Figure S27.Crude lipid to protein mass ratio for bench-scale experiments that replicated the conditions of the mix tank and PBRs.No significant change in biomass lipids content was demonstrated over the course of the experiments.Error bars represent minimum and maximum values from duplicate analyses.

Figure S28 .
Figure S28.Top: Biomass carbohydrate:protein ratio (by mass) in the full-scale Mix tank and PBR effluent between November 1, 2022 to February 14, 2023.Error bars represent minimum and maximum values from duplicate analyses.Bottom: Difference between the PBR and mix tank biomass carbohydrate:protein ratio between November 1, 2022 to February 14, 2023.Error bars represent the propagated error (Equation S1) of duplicate analyses.

/Analyzer Frequency of Data Sensor Location Calibration** , ***
*The PBR total volume was constant at 20,470 gal (full capacity) and was recorded at an average interval of 9 h.**Sensor and analyzer calibration times varied depending on the manufacturer's recommendations and the sensor performance.***Probes were cleaned three times per week by rinsing with tap water and gently wiping with a delicate task wipe (Kimwipes).

scale biomass composition trends S4.1. Long-term continuous monitoring of solids phosphorus content
Photosynthetically active radiation (PAR) 180-minute average at four locations on the PBRs (two upper fences and two lower fences) for the period between November 1, 2022 to February 14, 2023.PAR was measured by Model SQ-214 PAR sensors (Apogee Inc.).

Table S5 .
Criteria for sample selection for SEM-EDS analysis and key system operating conditions and performance during the sampling date.